Project Summary/Abstract Many important oncogenes in human cancers have normal coding sequences and amino acid structures, but become oncogenic due to genomic abnormalities that create strong transcriptional enhancers. Despite the central role of these aberrant enhancers in malignant transformation, insights into their mechanisms of action are based largely on associated chromosomal abnormalities, such as chromosomal translocations. Very recently, in studies of the oncogene TAL1 in T cell acute lymphoblastic leukemia (T-ALL), we discovered a new mechanism that promises to revise conventional perceptions of the role of aberrant transcriptional enhancers in cancer. We found that small, somatically acquired insertion mutations introduce binding motifs for the MYB transcription factor in a precise noncoding site, creating an aberrant transcriptional enhancer that drives high levels of TAL1 expression. This finding opens the opportunity to investigate how mutations and small insertions are formed in human cancer cells and how they create new enhancers to drive the expression of oncogenes critical for cell growth and survival. Such information will be vital on two fronts. (1) It will provide a conceptual framework for how normal enhancers regulate cell identity in the development of diverse tissues and (2) it will help to clarify the molecular mechanisms that distinguish normal from oncogenic enhancers, thus guiding the development of new targeted cancer treatments. My working hypothesis is that aberrant enhancers formed by somatic mutation in cancer cells are selected to provide the precise levels of oncogene expression needed to promote malignant transformation within a given cell lineage. My long-term goal is to experimentally dissect the specific DNA sequences, transcription factors, transcriptional adaptors and mediators, and epigenetic readers, writers and erasers that are required to form and sustain aberrant transcriptional enhancers capable of driving high levels of key oncogene expression in human cancer cells. To achieve this goal, I will focus initially on the analysis of human T-ALL and AML, and I propose to (i) identify diverse examples of enhancer mutations associated with the overexpression of key oncogenes in these leukemias; (ii) use CRISPR-cas9 mutagenesis to assess the dependence of the tumor cells on novel enhancer mutations for oncogene expression and malignant cell growth and survival; and (iii) define by RNAi which proteins in the epigenetic machinery (e.g., BRD4, CDK7, and CDK9) are required for the target oncogene to be overexpressed in tumor cells and the phenotypic consequences of downregulated expression. Finally, I will construct animal models in zebrafish and primary patient derived xenografts in mice to study molecular pathogenesis of aberrant transcriptional enhancers in vivo. With these animal models I will test strategies to eliminate these tumor cells with therapies designed to specifically target the mechanisms underlying aberrant transcriptional enhancers. Success in this research program will catalyze a paradigm shift in the understanding of oncogenic enhancers and their potential as candidates for targeted therapy.